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Whether synthetic genomes can power life has attracted broad interest in the synthetic biology field. Here, we report de novo synthesis of the largest eukaryotic chromosome thus far, synIV, a 1,454,621-bp yeast chromosome resulting from extensive genome streamlining and modification. We developed megachunk assembly combined with a hierarchical integration strategy, which significantly increased the accuracy and flexibility of synthetic chromosome construction. Besides the drastic sequence changes, we further manipulated the 3D structure of synIV to explore spatial gene regulation. Surprisingly, we found few gene expression changes, suggesting that positioning inside the yeast nucleoplasm plays a minor role in gene regulation. Lastly, we tethered synIV to the inner nuclear membrane via its hundreds of loxPsym sites and observed transcriptional repression of the entire chromosome, demonstrating chromosome-wide transcription manipulation without changing the DNA sequences. Our manipulation of the spatial structure of synIV sheds light on higher-order architectural design of the synthetic genomes., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc.; a founder of and consultant to Neochromosome, Inc; and a founder, Scientific Advisory Board (SAB) member of, and consultant to ReOpen Diagnostics, LLC. J.D.B. also serves or served on the SABs of the following: Logomix, Inc.; Sangamo, Inc.; Modern Meadow, Inc.; Rome Therapeutics, Inc.; Sample6, Inc.; Tessera Therapeutics, Inc.; and the Wyss Institute. Y.A. is a Founder and CSO of Logomix, Inc., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The Sc2.0 project is building a eukaryotic synthetic genome from scratch. A major milestone has been achieved with all individual Sc2.0 chromosomes assembled. Here, we describe the consolidation of multiple synthetic chromosomes using advanced endoreduplication intercrossing with tRNA expression cassettes to generate a strain with 6.5 synthetic chromosomes. The 3D chromosome organization and transcript isoform profiles were evaluated using Hi-C and long-read direct RNA sequencing. We developed CRISPR Directed Biallelic URA3-assisted Genome Scan, or "CRISPR D-BUGS," to map phenotypic variants caused by specific designer modifications, known as "bugs." We first fine-mapped a bug in synthetic chromosome II (synII) and then discovered a combinatorial interaction associated with synIII and synX, revealing an unexpected genetic interaction that links transcriptional regulation, inositol metabolism, and tRNA Ser CGA abundance. Finally, to expedite consolidation, we employed chromosome substitution to incorporate the largest chromosome (synIV), thereby consolidating >50% of the Sc2.0 genome in one strain., Competing Interests: Declaration of interests J.D.B. is a founder and director of CDI Labs, Inc., a founder of and consultant to Neochromosome, Inc., and a founder, SAB member of, and consultant to ReOpen Diagnostics, LLC and serves or served on the Scientific Advisory Board of the following: Sangamo, Inc., Logomix Inc., Modern Meadow, Inc., Rome Therapeutics, Inc., Sample6, Inc., Tessera Therapeutics, Inc., and the Wyss Institute., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

The characterization of cancer genomes has provided insight into somatically altered genes across tumors, transformed our understanding of cancer biology, and enabled tailoring of therapeutic strategies. However, the function of most cancer alleles remains mysterious, and many cancer features transcend their genomes. Consequently, tumor genomic characterization does not influence therapy for most patients. Approaches to understand the function and circuitry of cancer genes provide complementary approaches to elucidate both oncogene and non-oncogene dependencies. Emerging work indicates that the diversity of therapeutic targets engendered by non-oncogene dependencies is much larger than the list of recurrently mutated genes. Here we describe a framework for this expanded list of cancer targets, providing novel opportunities for clinical translation., Competing Interests: Declaration of interests G.B.M. is a science advisory board (SAB) member/consultant with AstraZeneca, Chrysallis Biotechnology, GSK, ImmunoMET, Ionis, Lilly, PDX Pharmaceuticals, Signalchem Lifesciences, Symphogen, Tarveda, Turbine, and Zentalis Pharmaceuticals; has stock/options/financial engagement with Catena Pharmaceuticals, ImmunoMet, SignalChem, and Tarveda; has licensed technology: HRD assay to Myriad Genetics and DSP patents with Nanostring; has sponsored research: Nanostring Center of Excellence and Ionis (Provision of tool compounds); and clinical trials support (funding or in kind) with AstraZeneca, Genentech, GSK, Lilly. S.L.S. is a shareholder and serves on the board of directors of Jnana Therapeutics; is a shareholder of Forma Therapeutics and Decibel Therapeutics; is a shareholder and advises Kojin Therapeutics, Kisbee Therapeutics, and Eikonizo Therapeutics; serves on the SABs of Eisai Co., Ono Pharma Foundation, Exo Therapeutics, and F-Prime Capital Partners; serves on the board of advisers of the Genomics Institute of the Novartis Research Foundation; and is a Novartis Faculty Scholar. W.C.H. is a consultant for ThermoFisher, Solasta, MPM Capital, iTeos, RAPPTA Therapeutics, Jubilant Therapeutics, and Paraxel and is a scientific founder and serves on the SAB for KSQ Therapeutics. J.S.B. is a founder and director of Neochromosome and holds an ownership equity interest in the company. A.C. is the founder and an equity holder in DarwinHealth, a company that has licensed algorithms for the analysis of regulatory networks and master regulator proteins from Columbia University. Columbia University is also an equity holder in DarwinHealth. C.J. Kemp is a founder and an equity holder in SEngine Precision Medicine, a company that harnesses 3D organoid technology and AI for more effective treatment options and accelerated drug development. W.K. and P.T. receive research support from Pfizer Oncology. W.A.W. is co-founder of StemSynergy Therapeutics. B.J.D. is an SAB member of Aileron Therapeutics, Therapy Architects (ALLCRON), Cepheid, Vivid Biosciences, Celgene, RUNX1 Research Program, Novartis, Gilead Sciences (inactive), Monojul (inactive); is an SAB member and holds stock in Aptose Biosciences, Blueprint Medicines, EnLiven Therapeutics, Iterion Therapeutics, Third Coast Therapeutics, GRAIL (SAB inactive); is a scientific founder of MolecularMD (inactive, acquired by ICON); is a member of the board of directors holds stock in Amgen; is a member of the board of directors of Burroughs Wellcome Fund and CureOne; is a member of the Joint Steering Committee of Beat AML LLS; is a founder of VB Therapeutics; has a sponsored research agreement for EnLiven Therapeutics; has clinical trial funding from Novartis, Bristol-Myers Squibb, Pfizer; and collects royalties from patent 6958335 (Novartis) and OHSU and Merck and one CytoImage exclusive license., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Nucleosome structural integrity underlies the regulation of DNA metabolism and transcription. Using a synthetic approach, a versatile library of 486 systematic histone H3 and H4 substitution and deletion mutants that probes the contribution of each residue to nucleosome function was generated in Saccharomyces cerevisiae. We probed fitness contributions of each residue to perturbations of chromosome integrity and transcription, mapping global patterns of chemical sensitivities and requirements for transcriptional silencing onto the nucleosome surface. Each histone mutant was tagged with unique molecular barcodes, facilitating identification of histone mutant pools through barcode amplification, labeling, and TAG microarray hybridization. Barcodes were used to score complex phenotypes such as competitive fitness in a chemostat, DNA repair proficiency, and synthetic genetic interactions, revealing new functions for distinct histone residues and new interdependencies among nucleosome components and their modifiers.

A network governing DNA integrity was identified in yeast by a global genetic analysis of synthetic fitness or lethality defect (SFL) interactions. Within this network, 16 functional modules or minipathways were defined based on patterns of global SFL interactions. Modules or genes involved in DNA replication, DNA-replication checkpoint (DRC) signaling, and oxidative stress response were identified as the major guardians against lethal spontaneous DNA damage, efficient repair of which requires the functions of the DNA-damage checkpoint signaling and multiple DNA-repair pathways. This genome-wide genetic interaction network also identified novel components (DIA2, NPT1, HST3, HST4, and the CSM1 module) that potentially contribute to mitotic DNA replication and genomic stability and revealed novel functions of well-studied genes (the CTF18 module) in DRC signaling. This network will guide more detailed characterization of mechanisms governing DNA integrity in yeast and other organisms.

Study of mutant phenotypes is a fundamental method for understanding gene function. The construction of a near-complete collection of yeast knockouts (YKO) and the unique molecular barcodes (or TAGs) that identify each strain has enabled quantitative functional profiling of Saccharomyces cerevisiae. By using these TAGs and the SGA reporter, MFA1pr-HIS3, which facilitates conversion of heterozygous diploid YKO strains into haploid mutants, we have developed a set of highly efficient microarray-based techniques, collectively referred as dSLAM (diploid-based synthetic lethality analysis on microarrays), to probe genome-wide gene-chemical and gene-gene interactions. Direct comparison revealed that these techniques are more robust than existing methods in functional profiling of the yeast genome. Widespread application of these tools will elucidate a comprehensive yeast genetic network.